JPH07198718A - Measurement of cardiac muscle troponin t using specific antibody for cardiac muscle troponin t - Google Patents

Abstract

PURPOSE: To monitor a duration for absolute diagnostic sensitivity to myocardial infarction and other myocardial damage by incarnating blood serum or blood plasma sample together with at least one antibody against myocardium toroponin T and toroponin T or antibody joint partner B. CONSTITUTION: Blood serum or blood plasma sample together with at least one antibody against myocardium toroponin T and toroponin T or antibody joint partner B is incarnated to label either of the antibody against toroponin T or the joint partner B with measurement allowable radicals. Immunological complex containing the measurement allowable radicals produced in such a way is isolated and measurement allowable radicals in the isolated phase or in the remaining phase are measured. The joint partner B should be jointed to the antibody against toroponin T or toroponin T. The joint partner B may be similar material which is jointed to the second antibody against toroponin T or Homo sapiens or animal inherent toroponin T or antibody.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for measuring cardiac troponin T using a specific antibody against cardiac troponin T,
In particular, it relates to the use of the method in determining myocardial necrosis.

[0002]

BACKGROUND ART Myofibrils of striated muscle are composed of two types of protein filaments that act in cooperation with each other, a thick filament has myosin as a main component, and a thin filament has actin, tropomyosin and troponin. Contains. Troponin, which is a regulatory structural protein, aggregates in cells to form a complex and forms three kinds of different proteins: troponin C (molecular weight 18000) that binds to calcium ion, and troponin I (subunit that binds to actin). It has a molecular weight of 24,000) and troponin T (molecular weight of 37,000) complexed with tropomyosin. This common nomenclature has historical reasons since the complex was first purified as such, considered a single protein, and named troponin. Later, it was proved that troponin was actually composed of three different proteins, but the positional relationship between each protein on the thin filament of the contractile organ,
And because of their coordination of regulation of muscle contraction,
This nomenclature was retained. However, they are three different proteins functionally related to other proteins of contractile organs such as myosin or actin, but with different amino acid sequences.

After a long period of severe ischemia or muscle cell necrosis, troponin I reaches the plasma and therefore troponin I
Is a parameter of such a disease and can be used for diagnosis and monitoring in the same manner as known infarction enzymes CK, CK-MB, GOT and LDH.

However, it has been proved that the measurement of CK and CK-MB is not specific to infarction and is only increased in serum 80 to 90 hours after infarction. Also, GOT and LDH are not specific to myocardium because increased amounts are also found in the blood in many other diseases.

The disadvantages of measuring cardiac troponin I are usually
The serum already contains troponin I at a concentration of 10 ng / ml [B. Cummins, Journal of Molecular and Cellular Cardiology (J. Mol. Cell. Card.). 19 (1987), 9
99-1010 and B. Cummins,
Clinical and Investigative Medicine (Clin. Invest.) 113 (1987) 1333-13
44]. Therefore, a two-phase serum concentration occurs in intramural infarction. On average, troponin I increased from 4 to 168 hours after the onset of pain in 37 patients with acute intramural infarction. Similar results were obtained in animal models. Thus, for troponin I, it is considered that the 10th to 50th hours after the occurrence of infarction becomes the time of absolute diagnostic sensitivity. That is, apart from sensitivity limitations, clinically significant monitoring of troponin I for 10 days or more after infarct development cannot be performed by measuring troponin I because of changes in serum levels of troponin I.

[0006]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to overcome these drawbacks and to enable a measurement method for myocardial infarction and other injuries to the myocardium for at least 150 hours (persistence of absolute diagnostic sensitivity). Is to provide.

[0007]

The object of the present invention is to provide a serum or plasma sample together with at least one antibody against cardiac troponin T (hereinafter also simply referred to as troponin T) and troponin T or a binding partner B of the antibody. Incubation, whereby either the antibody to troponin T or the binding partner B is labeled with a measurable group and the immunological complex containing the measurable group thus produced is isolated and the isolated phase or Cardiac Troponin T, characterized by measuring said measurable group in the remaining phase
By using troponin T derived from the sample as a measure of myocardial necrosis, it is possible to determine myocardial necrosis by an immunoassay.

The binding partner B must bind an antibody against troponin T or troponin T. The binding partner B may be, for example, a second antibody against troponin T or troponin T of human or animal origin, or their analogues bound by an antibody.

The sample is preferably incubated with a conjugate of an antibody against troponin T and another antibody against troponin T with a measurable group, the immunological complex formed being phase separated. And measure the measurable group in either phase.

Furthermore, the sample is incubated with an antibody against troponin T and a conjugate of troponin T and a measurable group, the immunological complex formed is isolated by phase separation and in either phase. It is preferred to measure the measurable group.

Surprisingly, in the measurement of myocardial necrosis (for example, due to myocardial infarction, ischemia or angina), troponin T immunoassay is more preferable than CK and CK.
It was found that a significantly higher sensitivity was obtained than by measuring other parameters like MB, GOT, LDH or Troponin I. As confirmed by the inventors, the reason for this is that unlike other proteins of contractile organs it does not measure serum concentrations of Troponin T up to the detection limit of the test (0.25 ng / ml). Is.

This is very surprising from the fact that a serum concentration similar to that of troponin I is expected for troponin T from the functional relationship between troponins. Furthermore, the serum concentration curve of troponin T shows that, for example, in intramural infarction, troponin I
Curve is significantly different. Unlike troponin I, the time course curves are now three-phase instead of two-phase, and troponin T has been found to increase on average up to 300 hours after the onset of pain. Absolute diagnostic sensitivity time is 6
It lasts from time to 195 hours. Thus, the time for absolute diagnostic sensitivity is almost four times that known for troponin I.

Radioimmunoassay, enzyme immunoassay,
All conventional immunoassays, such as fluorescent immunoassays,
Basically, it is suitable for the immunoassay method of the present invention. In addition, variations on these methods (eg, competitive immunoassay, I
EMA method) is applied. The sandwich test has proven to be particularly effective in determining Troponin T. This test method uses an immobilized antibody to troponin T and a conjugate of an antibody to troponin T and a measurable group. The details of the various variants of this test method and the performance of these methods are well described in the literature. However, for example, German patent application D
E-A3834766 and / or DE-A3822
Other immunological assays such as those described in 750 can also use the antibodies of the invention.

In the present invention, the antibody is a complete antibody,
Chimeric antibodies, bivalent antibodies and fragments thereof are included. The antibody to troponin T used may be polyclonal or monoclonal. Preferably, monoclonal antibodies are used.

In the sandwich test, in a preferred embodiment, at least two antibodies against troponin T are incubated with the sample solution. by this,
The first antibody mediates binding to the solid phase. To this end, the antibody may be bound to the solid phase either directly or via a spacer, or may be immobilized only after it is present in soluble form and an immunological reaction has taken place. The second antibody can be directly labeled with certain groups or attached to groups that can be measured by functional linkage. To this end, the antibody can bind to one of the specific binding pairs and the measurable group can bind to the other of the specific binding pairs. A complex containing the second antibody and the measurable group is formed during the reaction. The binding (immobilization) of the first antibody to the carrier is performed by adsorption, chemical binding or functional binding via a specific binding pair according to known methods. In this case, one of the binding pairs is immobilized while the other chemically binds to the antibody. The antibody can then be immobilized via this binding pair before or during the immunoassay reaction. Examples of such binding pairs include biotin streptavidin / avidin, hapten-antibodies, antigen-antibodies, sugar-lectins, hapten-binding proteins.

The competition test is a further preferred variant of the test. In this case, an antibody to Troponin T, which is immobilized before or during the measurement, and a conjugate of Troponin T and the measurable group are used.

Polystyrene is used as a material for a carrier for immobilizing an antibody or troponin T according to the present invention, for example, a tube, a plastic cuvette, a microtitration plate, beads or a plastic microcarrier.
Vinyl polymer, polypropylene, polycarbonate,
Materials such as polysaccharides, silicones, gums or treated glass can be used [eg.
ET Maggio, "Enzyme Immunoassay", CCA Press, Florida (1980), especially pages 175-178; E.
P-A-063064; Bioengineering (Bioe
ngineering 16 (1974), 997-1003; CJ Senderson and D.V. Wilson, Immunology.
(Immunology) 20 (1971), 1061-1065]. In particular, avidin- or streptavidin-coated carrier materials, in particular polystyrene, are used, preferably prepared according to the method described in EP-A-0269092. Similarly, binding partner B comprises either Troponin T or an analogue thereof or a monoclonal antibody capable of specifically binding Troponin T,
Be labeled. The usual reagents for the particular assay method are suitable for labeling. Thus, radioimmunoassays use radioisotopes such as ⁵⁷ Co for labeling. All enzymes commonly used in enzyme-immunoassays are suitable, eg peroxidase or β
-Galactosidase is suitable. Conventional fluorescent groups are suitable as labels for fluorescent immunoassays. Details of these various test methods and variations of the methods are known. The binding of Troponin T or antibody to the label can be done in a manner similar to binding to the solid phase, ie covalently or via a specific binding pair.

The antibody or troponin T can be bound to one of the above binding partners, for example via carbodiimide and hydroxysuccinimide, according to known methods.
Make a covalent bond.

Peroxidase (POD) as an enzyme label
Is preferably used.

One example of the antibody used in the present invention is
A polyclonal antibody capable of specifically binding to cardiac troponin T, which has less than 5% cross-reactivity with human skeletal muscle troponin T and less than 2% cross-reactivity with troponin I and other myofibrillar proteins. To produce this antibody, test animals, preferably sheep, are immunized with human cardiac troponin T and adjuvant at least 4 times every 4-6 weeks over a period of several months (preferably 4-6 months) and live serum is used. Is isolated and it is purified by immunoadsorption.

Further, another example of the antibody used in the present invention has less than 5% cross-reactivity with human skeletal muscle troponin T and less than 2% cross-reactivity with troponin I and other myofibrils. It is a monoclonal antibody that can specifically bind to cardiac troponin T. To produce this antibody, test animals, preferably inbred mice, were immunized intraperitoneally with human cardiac troponin T and adjuvant at least 4 times every 4 to 6 weeks for several months, followed by B lympholysis. Spheres are isolated, fused with a permanent myeloma cell line (cell line), clones isolated, and antibodies isolated from them.

Aluminum hydroxide and Bordatella pertussis or Freund's adjuvant are preferably used as adjuvants.

The well-known Koehler and Milstein [Nature 256 (1
975), 495-497], and the primary culture of the hybrid cells obtained during the fusion is performed in a conventional manner, for example using a commercially available cell sorter or by "limiting dilution". To clone. These cultures were further used to isolate isolated cardiac troponin T
And positively react with cardiac troponin T in the serum of the patient and negatively react with troponin T obtained from skeletal muscle.
The hybridoma cell line thus obtained produces the monoclonal antibody of the present invention. These cell lines can be cultured and the monoclonal antibodies produced therefrom can be isolated according to known methods.

As an example of the hybridoma cell line obtained by this method, clone 7.1 A12.2-22 (EC
ACC 89060901) and clone 8.1 F.
6.7-2 (ECACC 89030308). The cell line is the European Collection of Animal Cell Cultures.
Deposited under the respective numbers quoted at tion of Animal Cell Cultures (UK).

The polyclonal or monoclonal antibody thus obtained has low cross-reactivity to human skeletal muscle troponin T, less than 5%, preferably less than 2%, and troponin I and other muscles. Distinguished by less than 2% cross-reactivity with fibril proteins.

These polyclonal and monoclonal antibodies are particularly suitable for the specific measurement of myocardial necrosis in a sample such as serum or plasma. The antibodies can be used as is for these assays or as chimeric antibodies or fragments thereof, eg Fab fragments with corresponding immunological properties. Thus, the term "antibody" includes whole antibodies and fragments thereof.

[0027]

The present invention will be described below with reference to Reference Examples, Examples and the accompanying drawings, but the present invention is not limited thereto. Percent means% by weight.

Reference Example 1 Isolation of Troponin T Buffer 1: 0.05 mol / l Tris / HCl, pH 7.
0 0.05 mol / l KCl 10 ml / l Triton X 100 2 mmol / l EDTA 0.5 mmol / l DTT (dithiothreitol) 0.02% Sodium azide (w / v) 2 mmol / l PMSF (Phenylmethylsulfonyl fluoride) 5 mmol / l Aminocaproic acid 2.5 ml Trasylol / l 0.2 mg Pepstatin / l

Buffer 2: 0.05 mol / l Tris / HCl, pH 7.0 1 mol / l KCl 5 mol / l urea 2 mmol / l EDTA 0.02% sodium azide (w / v)

Buffer 3: 5 mmol / l potassium phosphate, pH 7.3 2 mol / l urea 0.6 mol / l KCl 0.5 mmol / l DTT 0.02% sodium azide (w / v)

Buffer 4: 0.05 mol / l Tris / HCl, pH 7.8 6 mol / l urea 0.01 mol / l NaCl 2 mmol / l EDTA 0.5 mmol / l DTT 0.02% azide Sodium (w / v)

Tissue (left ventricle excluding valves and blood vessels) 30
0 g was chopped into small pieces and homogenized intermittently with a mixer in 4 volumes of buffer 1 for up to 1 minute at 4 ° C. The insoluble components were centrifuged at 27,500 g for 15 minutes at 4 ° C. and the pellet was washed with 1 liter of buffer 1 until the supernatant was completely clear.

This pellet is mixed with 5 to 6 volumes of buffer solution 2
And stirred at 4 ° C. for 3-4 hours. 15 at 4 ℃
Centrifuged at 27500 g for minutes.

5 mmol / l ATP was added to the supernatant (final concentration after addition of ammonium sulphate), saturated aqueous ammonium sulphate solution at 4 ° C./2 mmol / l ED.
35% ammonium sulfate was made by addition of TA.
The ammonium sulfate solution was previously neutralized with KOH.
Incubate overnight at 4 ° C. with slow agitation. The precipitate was then centrifuged at 27500 g for 20 minutes at 4 ° C.

Solid ammonium sulfate was added to the supernatant to make it 45% saturated with ammonium sulfate, stirred at 4 ° C. for 45 minutes, and then centrifuged. The 35-45% ammonium sulphate precipitate contained Troponin T and was further processed.
The pellet was taken up in 25 ml of buffer 3 (OD 280 nm is 1.5-2) and dialyzed against buffer 3 at 4 ° C., during which the buffer was changed at least twice.

The dialyzed solution was applied to a hydroxylapatite column [Biogel HTP (Bioge).
1 HTP), about 25 mg protein / 50 ml column volume, flow rate: 1 column volume / hour, and then rewashed with 4 to 5 column volumes times buffer solution 3 and 5 mmol / liter to 55 mmol in buffer solution 3. / Liter of a potassium phosphate gradient solution was eluted within 20 hours at 1 column volume times / hour.
Fractions containing Troponin T were measured using SDS-PAGE (gel gradient 5-25%), pooled and dialyzed against buffer 4.

The dialyzed solution was applied to a column [DEAE-Servacel SS].
23, column volume 20-30 ml, flow rate: 1 column volume /
Hours] Rewash with 3 column volumes of buffer 4
Medium N to 0.01 mol / liter to 0.25 mol / liter
The aCl gradient eluted at 1 column volume / hour within 20 hours. Troponin I was found in the eluate. Troponin T and Troponin C were eluted separately. SDS-P
Fractions containing troponin T were collected according to AGE,
Redialyze against Buffer 4 and -20 ° C for later use
Frozen in. From 300g muscle to Troponin T about 6-8m
g was obtained.

Reference Example 2 Isolation of Monoclonal Antibody to Human Troponin T Balb / c mice aged 8 to 12 weeks were intraperitoneally injected with 100 μg of Troponin T (isolated from human myocardium according to Reference Example 1) together with Freund's complete adjuvant. Immunization was performed by internal administration. 6 weeks later, 3 times every 4 weeks, 50 μg each
Was immunized with aluminum troponin T, adsorbed on aluminum hydroxide, and Bordetella pertussis was intraperitoneally administered. One week after the final immunization, blood samples were taken and the antibody titers in the serum of the test animals were measured. If the immunity is positive, fusion is performed. They were treated with troponin T10 in PBS (phosphate buffered saline) 4, 3 and 2 days before fusion, respectively.
Re-immunization was carried out by intravenous administration at 0 μg.

1 × 10 of immunized mice for fusion
⁸ Spleen cells were transformed into Galfre [Enzymology (E
nzymology), 73, 1981, p.3].
× 10 ⁷ myeloma cells (P3x63Ag8-653, ATC
C-CRL 8375) and then centrifuged for 10 minutes (300 g, 4 ° C). The cells were washed with serum-free medium and centrifuged again at 400g. The supernatant is aspirated and the cell pellet is subjected to 50% PEG solution [molecular weight 4000, Merck.
1 ml of Merck Company) was added. Then, within 15 minutes at room temperature, RPMI1640 medium without fetal calf serum (FCS) [RPMI = Rosewell Parker Memory Institute (Rosewe
ll Parker Memory Institute)] and slowly diluted to 20 ml. Then, the cell suspension was centrifuged at 400 g for 10 minutes, and the cell precipitate was filtered with a selective medium (RPMI).
1640, 10% FCS, 100 mmol / liter
Hypoxanthine, 1 mg / ml azaserine). For growth factors, HECS [Human Endothelial Culture Super
natant)] [Costar Company
y), No. 6110].

Fused cells were seeded on 24-well plates (Nunc Company) at 5 × 10 ⁴ cells per well. Clone growth was visible to the naked eye after 7-10 days. According to the ELISA method described in Reference Example 3,
The culture supernatant of the primary culture was tested. Primary culture containing antigen-specific antibody was added to 96-well cell culture plate
Further cloning was performed using a fluorescence activated cell sorter on (Nank Company). HE as an alternative to supply cells
CS was used (see above).

Thus, the hybridoma cell line clones 7.1A12.2-22 and 8.1F6.7-
Both of the two could be isolated. These are trustees E
CACC [European Collection of Animal Cell Cultures]
nimal Cell Cultures)] under the following accession numbers: ECACC 89060901 (= clone 7.1A12.2-22) and ECACC 89030.
308 (= clone 8.1F6.7-2).

To obtain ascites, Pristan
Balb / c mice pre-treated with 0.5 ml once or twice were injected ip with 5 × 10 ⁶ hybridoma cells. Two
Ascites could be obtained from the abdomen of the mice after ˜3 weeks.
Antibodies could be isolated from this ascites by conventional methods. These monoclonal antibodies are human cardiac troponin T
Troponin T obtained from skeletal muscle that is specifically directed to
There was no or little cross-reactivity with. Next, these are MAB 7.1A12.2-2.
2 (from clone 7.1A12.2-22) or MAB
It was named 8.1F6.7-2 (derived from clone 8.1F6.7-2). Both monoclonal antibodies are subclass IgG
1 / belongs to kappa.

Reference Example 3 Screening Test of Antibodies to Human Cardiac Troponin T In order to determine the presence and specificity of antibodies to troponin T in the serum of immunized mice, the test was carried out in a culture suspension of hybrid cells or ascites. E as a basis
LISA method was used. That is, coating buffer (0.2 mol / liter sodium carbonate / sodium bicarbonate, pH
9.3-9.5) at room temperature for 1 hour, 10 μg / ml polyclonal antibody against human troponin T from sheep (I
microtiter plates were coated with gG) (purified by immunoadsorption; not cross-reactive with skeletal muscle troponin T, but with cardiac troponin T; prepared according to Reference Example 6). 20 with 0.9% sodium chloride solution and 1% bovine serum albumin
Recoated for minutes. After that, it was washed with a washing buffer (0.9% sodium chloride solution). Incubation of antigen, purified 1 μg / ml human troponin T or "native" troponin T, ie infarct serum (diluted 1: 2), using 100 μl per well with shaking at room temperature for 1 hour. It was It was then washed twice with wash buffer. 1 per well with shaking at room temperature for 1 hour
Samples were incubated with 00 μl. Then, it was washed twice with the washing liquid. Then, at room temperature for another hour with shaking, 25 mU PAB per well.
Incubated with 100 μl <M-Fcg> S-Fab (IS) -peroxidase conjugate. Here, PAB <M-Fcg> S-Fab (IS) is a Fab fragment of a sheep-derived polyclonal anti-mouse Fcγ antibody purified by immunoadsorption. After the washing step using the washing buffer, the difference in the absorbance at 405 nm was measured using a conventional method (eg, ABTS at room temperature for 30 minutes).
Peroxidase activity was measured).

Reference Example 4 Measurement of cross-reactivity with human skeletal muscle troponin T The method of the present invention was carried out as described in Reference Example 3. First, the reactivity of cardiac troponin T was measured. The antigen to be tested for cross-reactivity (skeletal muscle troponin T) was then added to each monoclonal antibody in increasing concentrations. The cross-reactivity was then calculated according to the following formula:

[0045]

[Equation 1]

Reference Example 5 Measurement of Epitope Specificity: 10 μg / ml against Fcg region of mouse antibody in 0.2 mmol / liter carbonate buffer (pH 9.6) at room temperature for 1 hour or at 4 ° C. overnight. A microtiter plate was coated with sheep polyclonal antibody. It is then recoated with incubation buffer (0.9% sodium chloride solution and 1% bovine serum albumin) for 20 minutes, then wash buffer [0.9% sodium chloride solution, 0.05
% Tween 20]. Then, incubation buffer (0.1% bovine serum albumin containing 0.1%) was used.
10 μg / ml monoclonal antibody (MAB 7.1A 12.2-22, MAB 1) 100 in 9% sodium chloride solution)
μl was added and incubated at room temperature for 1 hour with shaking. A second monoclonal antibody (MAB 8.1F6.7- present as a peroxidase conjugate).
2, MAB 2) at room temperature in solution (100 mU / ml),
Preincubation with antigen (1 μg / ml cardiac troponin T).

After incubating the plate with MAB 1, excess antibody was removed by washing. The plates were then recoated with 1% mouse normal serum in incubation buffer. 100 μl of pre-incubated Troponin T / MAB 2 peroxidase complex was placed on the plate and incubated at room temperature for 1 hour with shaking. The bound peroxidase activity was made visible with the naked eye using ABTS as a substrate.
Recognizing that MAB 2 is the same or overlapping epitope as MAB 1, no pair is formed between MAB 1 / troponin T / MAB 2, resulting in no substrate reaction. The results obtained showed that both monoclonal antibodies 7.1A12.2-22 and 8.1F6.7-2 are directed against different epitopes of the cardiac troponin T antigen.

Example 1 Enzyme for measurement of troponin T by ELISA method-
Immunoassay Reagent 1 1.25 μg / ml Biotinylated MAB 7.1A12.2-
22 [JH Peters et al.
al.), Monoklonale Anti-Kelpel (Monoklonale)
Antikoerper), Springer Berlag (Springer)
Verlag), Berlin, 1985, pages 209-212.
Page] 10 mmol / liter citrate buffer 47 mmol / liter phosphate buffer, pH 6.3 50 mU / ml peroxidase and monoclonal antibody 8.
Conjugate with 1F6.7-2 [W nap
(W.Knapp), K. Kolubar, G.
Immunofluorescence and Related Staining Technics, edited by G. Wick
(Immunofluorescence and Relatated Staining Techn
iques (p.215-p.224, Elsvia)
evier) / MB Wilson (North Holland, Amsterdam)
son), P.K.Nakane (1987)
The value of activity is related to peroxidase, prepared by a method similar to that of.

Reagent 2 100 mmol / l phosphate-citrate buffer, pH 4.4 3.2 mmol / l sodium perborate 1.9 mmol / l ABTS (2,2'-azino-
Di- [ethylbenzothiazoline-sulfonate (6)]).

As a sample, 3, 5, 10 or 25 ng /
Human serum supplemented with ml troponin T was used. Polystyrene tubes coated with streptavidin (EP-A-02
(Prepared according to 69092).

Assay: 0.2 ml sample was incubated with 1 ml reagent 1 in a tube at 20-25 ° C. for 60 minutes. It was aspirated and washed twice with tap water. Next, reagent 2
And incubate at 20-25 ° C for 30 minutes,
Absorbance at 420 nm was measured with a photometer. using this method,
A standard curve (Fig. 1) is obtained, by means of which the troponin T concentration of the patient sample can be measured.

Reference Example 6 Isolation and Purification of Polyclonal Antibody against Troponin T First, 0.1 m in Freund's complete adjuvant (CFA) was prepared.
The sheep were immunized with 55 μl of a solution of g / ml human troponin T. Furthermore, immunization was performed on the 7th, 14th and 30th days. Next, immunization was performed every 30 days. 28 μl of a 0.05 mg / ml troponin T solution in CFA was used for each subsequent immunization. After 6 months, raw serum was obtained. 15 g of Aerosil [manufacturer: Degussa] was added to 1 liter of raw serum, and the mixture was stirred at room temperature for 1 hour and centrifuged. Then, 1.7 mol / liter ammonium sulfate was added to the supernatant, and the mixture was slowly stirred at room temperature for 2 hours. Then, the precipitate is centrifuged and the dialysis buffer is added.
It was homogenized in 0.2 liter (15 mmol / liter potassium phosphate, 50 mmol / liter sodium chloride, pH 7.0) and dialyzed against 10 liters of dialysis buffer 4 times at 4 ° C. After centrifugation, the dialyzed product was purified on DE52 cellulose using dialysis buffer as eluent.

The eluate was supplemented and at a protein concentration of 15 mg / ml, 50 mmol / liter potassium phosphate, pH
7.5, 150 mmol / liter sodium chloride (P
BS), 0.1% sodium azide, and passed through a column packed with skeletal troponin T immunosorbent at room temperature to remove antibodies that cross-react with skeletal troponin T. The cross-reactivity of the antibody that did not bind to the skeletal troponin T adsorbent in the antibody fraction was tested by the ELISA method of Reference Example 4, and this operation was repeated until the desired cross-reactivity was obtained. Then, the antibody fraction was loaded on a cardiac troponin T immunoadsorbent column, and the protein was washed off with PBS / 0.1% azide. After that, it was eluted with 1 mol / liter propionic acid. The eluate was dialyzed against 15 mmol / liter potassium phosphate and 50 mmol / liter sodium chloride (pH 7.0). Spherodyl
rosil) [Rhone Poulenc XOC0
05] was washed with 15% nitric acid and water, dried, and then
Spherodyl- using 10% (v / v) 3- (triethoxysilyl) propylamine in DMSO overnight at 85 ° C.
A troponin T immunoadsorbent was prepared by converting to NH ₂ . After this reaction, it was washed with DMSO and isopropanol, and the adsorbent was dried at 50 ° C.

Spherodil-NH ₂ was mixed with a 10% glutardialdehyde solution (pH 3.7) and heated at 55 ° C. for 2 hours. Then, the suspension was filtered through a glass filter under vacuum. Then, it is washed with double-distilled water 7 times by volume of spherodil, and 5 times by volume 10 mmol / liter.
It was washed again with potassium phosphate, pH 8.0 / 0.1 mol / liter sodium chloride. A round bottom flask was then charged with 10 mg of troponin T per ml of spherodil in 10 mmol / liter potassium phosphate, pH 8 / 0.1 mol / liter sodium chloride in a volume of about 50% of the spherodil used. The flask was spun at room temperature overnight on a rotary evaporator. After filtration, spherodil was treated with 0.9% NaCl solution and ethanolamine solution,
It was washed again several times. Then, 3 parts by volume of ethanolamine solution was added, and the mixture was incubated at room temperature for 1 hour.
After restarting the filtration, it was washed again with a NaCl solution. After that, the immunoadsorbent was adjusted to pH 7.5 with PBS and PBS /
Equilibrated with sodium azide. Stored at 4 ° C.

Example 2 Determination of Myocardial Necrosis According to Example 1, 37 with unstable angina symptoms.
An enzyme-immunoassay for troponin T measurement was performed on body patient sera. 13 of these patients (30%)
There was a significant increase in troponin T in. This indicated that the measurement of Troponin T had a clearly higher sensitivity for the detection of small infarcts compared to the Troponin I test. In intramural infarction, the serum concentration curve of Troponin T differs significantly from that of Troponin I. This is shown in Figure 2, which shows the mean serum concentration curve of 52 patients with intramural myocardial infarction. In this infarct group, the average troponin T is
It was found to increase> 300 hours after the onset of pain.
The time for absolute diagnostic sensitivity in this group of patients is
It is from the 6th hour to the 195th hour.

The infarct size is very different (Q-
Wave and non-Q-wave AMI) In a larger group of patients, the time for absolute diagnostic sensitivity of troponin T was
It was 12 to 140 hours after the onset of pain. Troponin T release is characteristic of cytosolic and structurally bound marker proteins. In reperfused AMI, a prominent troponin T peak was seen on day 1, which was absent in non-reperfused AMI (Figure 3). Perfusion-dependent changes in early troponin T release can be used to predict the effect of thrombolytic therapy without invasion. No similar results were seen with other marker proteins including troponin I.

[0057]

INDUSTRIAL APPLICABILITY According to the present invention, an immunoassay for cardiac troponin T capable of maintaining absolute diagnostic sensitivity for at least 150 hours in myocardial infarction and other damages to the myocardium can be provided, and is effective in determining myocardial necrosis. Available for

[Brief description of drawings]

FIG. 1 is a graph showing a standard curve for the Troponin T assay.

FIG. 2 is a graph showing the mean serum concentration of troponin T in 52 patients with intramural myocardial infarction.

FIG. 3: Twenty patients (ER) with <3.5 h infarct reperfusion after pain onset, 20 patients (LR) with> 3.5 h of pain onset reperfusion, and non-reperfusion. FIG. 9 is a graph showing a smooth scan plot with locally weighted regression lines of the relative increase in serum troponin T concentration in 24 patients with perfusion infarction (PO). Note the marked early troponin T release in successfully reperfused infarcts.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical indication location C12N 15/02 C12P 21/08 9161-4B (C12P 21/08 C12R 1:91) (72) Invention Anelise Volga Germany 8132 Chutting, Kustermann Strasse 3 (72) Inventor Klaus Hallermeier Germany 8000 Munich-2, Ballstrasse 31 (72) Inventor Siegfried Roselle Germany 8120 No. 27, Ammerstrasse, Weilheim

Claims (3)

[Claims] 1. At least 1 for cardiac troponin T
Serum or plasma sample with one antibody and cardiac troponin T or binding partner B of said antibody,
Thereby, either the antibody to cardiac troponin T or the binding partner B is labeled with a measurable group and the immunological complex containing the measurable group thus produced is isolated and the isolated phase or the remaining phase A method for measuring cardiac troponin T, which comprises measuring the measurable group in the phase. 2. A monoclonal antibody to human cardiac troponin T, wherein the antibody has <2% cross-reactivity to troponin I and other myofibrillar proteins and <5% cross-reactivity to skeletal muscle troponin T, or The measuring method according to claim 1, which is a polyclonal antibody. 3. The measuring method according to claim 1, which is used for determination of myocardial necrosis.